Site-specific modification of amino acids and peptides by aldehyde-alkyne-amine coupling under ambient aqueous conditions

Article abstract of DOI:10.1021/ol301017q

A highly efficient method for the direct, site-specific functionalization of amino acids and peptides, under ambient conditions, is described. In aqueous, nearly solvent-free conditions, copper(I) chloride catalyzed the aldehyde-alkyne-amine (A³) coupling of amino acids to form dipropargylated products in moderate to excellent yields. The propargylamine functionality provides a convenient handle for further structural modifications, demonstrated by a subsequent one-pot deprotection and click reaction and a solution-phase peptide coupling.

Full text of DOI:10.1021/ol301017q

ORGANIC
LETTERS
2012
Vol. 14, No. 12
3000–3003
Site-Specific Modification of Amino Acids
and Peptides by AldehydeꢀAlkyneꢀ
Amine Coupling under Ambient
Aqueous Conditions
Nick Uhlig* and Chao-Jun Li*
Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montreal,
Quebec H3A0B8, Canada
nicholas.uhlig@mail.mcgill.ca; cj.li@mcgill.ca
Received April 18, 2012
ABSTRACT
A highly efficient method for the direct, site-specific functionalization of amino acids and peptides, under ambient conditions, is described. In
aqueous, nearly solvent-free conditions, copper(I) chloride catalyzed the aldehydeꢀalkyneꢀamine (A3) coupling of amino acids to form
dipropargylated products in moderate to excellent yields. The propargylamine functionality provides a convenient handle for further structural
modifications, demonstrated by a subsequent one-pot deprotection and “click” reaction and a solution-phase peptide coupling.
The selective and bio-orthogonal functionalization of
biomolecules is a highly desirable tool for chemists and
biologists alike. Peptides, in particular, are frequent targets
of modification, as a method for alteration of their chemi-
cal and pharmacological properties for use as therapeutic
agents.1 In the past century, this field has seen impressive
growth.2 From simple modifications of nucleophilic resi-
dues such as lysine and cysteine, there now exist methods
for functionalizing the carboxylic acid functionalities of
glutamic and aspartic acid,3 rare aromatic moieties such as
tryptophan,4 as well as tyrosine,5 phenylalanine,6 and
arginine residues.7 The site-selective C-functionalization
of glycine redisues in peptides has also been explored.8
New methods for the functionalization of N-terminal and
lysine side-chain amines have also been developed, most of
which rely on direct or reductive alkylation.9
The use of alkynes as a coupling partner for imines;an
intermediate in the reductive alkylation of amines with
aldehydes;allows an equivalent reductive alkylation of
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2004, 126, 15942. (b) Romanini, D. W.; Francis, M. B. Bioconjugate
Chem. 2008, 19, 153. (c) Tilley, S. D.; Francis, M. B. J. Am. Chem. Soc.
2006, 128, 1080.
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Fontana, A. Biochemistry 1998, 37, 13507. (b) Jungheim, L. N.;
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Huang, J.-S.; Wong, M.-K.; Che, C.-M. J. Am. Chem. Soc. 2012, 134,
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r
10.1021/ol301017q
Published on Web 05/30/2012
2012 American Chemical Society

amines while at the same time appending an alkyne;a
highly useful moiety in bioorthogonal chemistry (scheme 1).10
This three-component coupling is commonly referred to as
the alkyneꢀaldehydeꢀamine or A3 coupling.11 The A3
coupling has been accomplished with a very broad range of
transition metals, including silver,12 gold,13ruthenium/
copper,14 cobalt,15 copper,16 indium,17 iridium,18 and iron.19
We envisioned that the A3 coupling could act as a process
that combines the selectivity of reductive alkylation with the
utility of alkynylation in a single reaction, preferably under
mild conditions and with inexpensive catalysts.
Herein, we report such a method for the site- selective
modification of peptides and amino acids via copper(I)-
catalyzed A3 coupling under ambient, aqueous conditions.
Initial studies began based on a five-component, ruthe-
nium/copper-catalyzed variety of the A3 coupling.14b The
reaction of glycine methyl ester 1a, with phenylacetylene
and formaldehyde toproduce amino acidderivative4awas
used as the testing model (Table 1).
Table 1. Catalyst and Condition Screening for the Reaction of
1a, 2a, and 3a To Create Propargylamine 4aa
solvent,
time
(h)
yieldc
(%)
entry
catalystb (%)
temp (°C)
1
2
RuCl3/CuBr (5/15)
RuCl3/CuBr (5/15)
RuCl3/CuBr (5/15)
CuOTf (10)
H2O, rt
48
96
48
48
48
48
48
48
48
48
48
48
18
18
18
18
43
61
H2O, rt
3
H2O, 40
55
4
MeCN, 60
MeCN, 60
MeCN, 60
MeCN., 60
MeCN, 60
MeCN, 60
MeCN, 60
H2O, 60d
H2O, 35d
H2O, 35d
H2O, 35d,e
H2O, rtd
47
5
CuOTf/bipy (10/10)
CuOTf/4,40-MeO-bipy
CuOTf/phen (10/10)
CuOTf/terpyr (10/10)
CuBr (10)
80
6
70
7
67
8
85
9
80
10
11
12
13
14
15
16
CuI (10)
95
CuI (10)
54
CuI (10)
42
CuCl (10)
>95
>95
78
Scheme 1. Direct Alkynylation of Free Amines Using the A3
Reaction Represents an Interesting Alternative to Alkylation
CuCl (10)
CuCl (10)
CuCl2ꢀH2O (10)
H2O, 35d
85
a Conditions: glycine methyl ester hydrochloride (0.2 mmol), for-
maldehyde (0.5 mmol, 37% in water), phenylacetylene (0.5 mmol), CuCl
(0.02 mmol), and NaHCO3 (0.2 mmol), 0.5 mL of solvent, under Ar.
b CuOTf = copper(I) triflate toluene complex; bipy = 2,20-bipyridine;
4,40-MeO-bipy = 4,40-dimethoxy-2,20-bipyridine; phen = 1,10-phenan-
throline; terpyr = 2,20:60,200-terpyridine. c Yields were determined by
NMR spectroscopy using mesitylene as an internal standard. d Reaction
was run with no additional solvent; only phenylacetylene and water from
the formaldehyde solution were present as liquids in the vessel. e Reac-
tion was run under air atmosphere.
Even without ruthenium, the use of terpyridine or
bipyridines with copper(I) triflate afforded good yields in
acetonitrile solvent (Table 1, entries 4ꢀ8). However, by
using only copper(I) chloride and neat conditions, a dra-
matic increase in yield was observed (entry 13).
Interestingly, CuCl2 provided yields nearly as high as
CuCl. The high effective concentration of these reactions
would allow reduction of the aqueous Cu2þ to the active
Cu1þ species by the alkyne (via oxidative dimerization) or
by methanol (present in the formaldehyde solution) to
occur quickly. The rapid formation of a canary-yellow
precipitate in these reactions, characteristic of polymeric
Cu(I)ꢀladderane complexes,20 supports this theory. Ex-
clusion of oxygen was not necessary for excellent yields
(entry 14) but precluded formation of small quantities of
the alkyne homocoupling product.
(10) For a recent review of alkyne chemistry in biological contexts,
see:Uhlig, N.; Li, C.-J. Chem. Sci. 2011, 2, 1241.
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Acta 2011, 44, 43. (c) Zani, L.; Bolm, C. Chem. Commun. 2006, 4263–
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2006, 8, 1529. (b) Wei, C.; Li, C.-J. J. Am. Chem. Soc. 2003, 125, 9584.
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Encouraged by this promising result, the scope of the
reaction was tested with a number of alkynes (Scheme 2).
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Org. Lett., Vol. 14, No. 12, 2012
3001

To our delight, every alkyne substrate tested produced
moderate to excellent yields of the desired products,
though alkynes containing highly electron-withdrawing
substituents gave less favorable results (4d, 4e). Aliphatic
alkynes also gave poorer yield (4f), though TMS-acetylene
gave excellent yield under the same conditions (4g).
Scheme 3. A3 Coupling of Amino Acids and Dipeptides with
Phenylacetylene and Formaldehydea
Scheme 2. A3 Coupling of Alkynes, Formaldehyde, and Glycine
Methyl Estera
a Reaction conditions: amino acid, sodium bicarbonate (1 equiv),
formaldehyde (2.5 equiv as 37% aqueous solution), phenylacetylene (2.5
equiv), copper(I) chloride (10 mol %), under argon, 35 °C, 18 h. Isolated
yields based on the amino acid. b Product was purified by preparatory
thin-layer chromatography. c Dihydrochloride salts were used; 5 equiv
each of formaldehyde and phenylacetylene and 2 equiv of NaHCO3 were
used. d No sodium bicarbonate was added.
a Reaction conditions: glycine methyl ester hydrochloride, sodium
bicarbonate (1 equiv), 37% formaldehyde (2.5 equiv as 37% aqueous
solution), alkyne (2.5 equiv), argon, 35 °C, 18 h. Isolated yields, based on
glycine methyl ester hydrochloride. b Product was purified by prepara-
tory thin-layer chromatography.
For the functionalization of various amino acids and
dipeptides, a large variety of functional groups (disulfides,
esters, alcohols, phenols, guanidines, secondary amides,
thioethers, and carboxylic acids) proved to be tolerated by
the reaction, and very little purification was required
(Scheme 3). In most cases, only evaporation of the excess
reagents under high vacuum was necessary to yield a pure
product. The exceptions to this were products 4e
(Scheme 2) and 5a, 5c, and 5i (Scheme 3). The reaction
of cystine dimethyl ester gave the desired tetrafunctiona-
lized dimer product in moderate yield but also yielded a
small amount of a possible monomer cyclized product.
Cysteine ethyl ester gave nearly quantitative yield of this
cyclized thiazolidine product 5b, the precursor to which is
known to be produced upon reaction of cysteine with
formaldehyde.21 Boc-protected tryptophan gave a similar
result, producing the interesting tetrahydrocarboline
Scheme 4. One-Pot Deprotection and “Click” Functionaliza-
tion of Dipropargylated Glycine Derivative 4g
derivative 5j. While several amino acids participate in this
side reaction (including tryptophan, cysteine, serine, as-
paragine, and histidine),20 it only occurs when said amino
acid is present at the N-terminus. Serine methyl ester gave
a somewhat decreased yield of the desired product 5i,
along with 6 mg of a second product which could not
be conclusively identified at this time, but may be the
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3002
Org. Lett., Vol. 14, No. 12, 2012

A one-pot deprotectionꢀCuAAC reaction was per-
formed on compound 4g using a silver-catalyzed deprotec-
tion of TMS-alkynes22 followed by direct addition of
benzyl azide and a copper catalyst to effect the CuAAC
reaction.23 This procedure afforded the bis-triazole pro-
duct 6a in excellent yield (Scheme 4).
Scheme 5. Boc-Protected Lysine Derivative 5f Can Be Easily
Deprotected or Subjected to Solution-Phase Peptide Couplings
To Produce Side-Chain Functionalized Lysine Derivatives (8a)
or Peptides (10a) in Excellent Yield
In addition, the excellent tolerance of sensitive groups
such as tert-butoxycarbonyl protecting groups suggested
the possibility of incorporating side-chain-functionalized
lysine residues into synthetic peptides. To this end, product
5g was converted to the activated pentafluorophenyl ester
8a, which was then used directly in a solution-phase
peptide coupling (Scheme 5) with L-alanine to yield the
N-protected dipeptide 10a in excellent yield and with no
epimerization of either stereocenter.24 Both this resulting
dipeptide and the original lysine derivative 5g could be
easily deprotected to their yield 10a and 7a, respectively
(Scheme 5).
In summary, we have developed a highly efficient method
for the direct functionalization of amino acids and peptides
under ambient, aqueous conditions via the A3 coupling,
catalyzed by copper(I) chloride. The reaction showed
tremendous scope, being compatible with thioethers,
ethers, secondary amides, hydroxyl groups,esters, car-
boxylic acids, guanidine, and;to a lesser extent;disulfide
groups, as well as a wide variety of alkynes. These results
indicate great potential for the use of the A3 reaction as a
method to modify and conjugate because of its simplicity,
low cost, tolerance of functional groups, exceedingly mild
conditions, and ease of transformation of its products into
further useful compounds. Studies aimed at reducing
concentration and application to other biomolecules are
ongoing.
oxazolidine product. The reaction was additionally tested
on several dipeptides to examine its potential use for the
direct functionalization of larger structures. Glycylglycine
ethyl ester, glycylleucine, and glycylserine all gave good to
excellent yields of the N-terminal difunctionalized pro-
ducts (5j, 5k, and 5l, respectively).
Due to ethynyltrimethylsilane’s excellent yield in the
functionalization of glycine methyl ester, it was envisioned
that this substrate combination could provide a convenient
route to the free alkyne-functionalized product, which
could in turn be functionalized using the copper(I)-cata-
lyzed azideꢀalkyne click (CuAAC) reaction.
Acknowledgment. We are grateful to the Canada Re-
search Chair (Tier 1) foundation (to C.-J.L.) and NSERC
for their support of our research. N.U. thanks Jacqueline
Yip (McGIll) for valuable assistance in the early stages of
the project.
Supporting Information Available.
Detailed experi-
mental procedures and physical and spectral data for all
new compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
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The authors declare no competing financial interests.
Org. Lett., Vol. 14, No. 12, 2012
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